6EXL

The Transcriptional Regulator PrfA from Listeria Monocytogenes in complex with a ring-fused 2-pyridone (MK206) - folded HTH motif

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ESRF BEAMLINE ID23-1
Synchrotron site	ESRF
Beamline	ID23-1
Temperature [K]	100
Detector technology	PIXEL
Collection date	2015-12-15
Detector	DECTRIS PILATUS 6M-F
Wavelength(s)	0.975
Spacegroup name	P 21 21 21
Unit cell lengths	47.749, 87.795, 116.183
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	43.900 - 1.900
R-factor	0.175
Rwork	0.173
R-free	0.22500
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	5f1r
RMSD bond length	0.021
RMSD bond angle	1.154
Data reduction software	XDS
Data scaling software	Aimless
Phasing software	PHASER
Refinement software	PHENIX ((1.10.1_2155: ???))

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	43.897	1.970
High resolution limit [Å]	1.900	1.900
Rmerge	0.089	0.783
Number of reflections	38994
<I/σ(I)>	13.4	2.5
Completeness [%]	99.4	97.9
Redundancy	7.2	6.9

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	5.5	291	Following cell lysis, the protein was extracted by Ni-NTA Superflow FF, followed by TEV protease cleavage in buffer (200 mM NaCl, 1 mM BME, 20 mM NaP (NaH2PO4+Na2HPO4, pH 7.1). Further purification included Ni-NTA, MonoS and Gelfiltration (in 200 mM NaCl and 20 mM NaP buffer pH 6.5). Purified PrfA was co-crystallized with complex (5 mol excess) using the hanging-drop vapor-diffusion technique. Crystals grew in 5 days after 2 microL of the protein solution (3.2-3.5 mg per ml PrfA, 200 mM NaCl, 20 mM NaP buffer, pH 6.5) was mixed with an equal volume of precipitant solution containing 20-24% PEG-4000, 17% isopropanol, 100 mM Na citrate pH 5.5 and allowed to equilibrate over a 1 ml solution of the precipitant in a Linbro plate (Hampton Research).